Candida albicans is the most prevalent human fungal pathogen and is an important cause of morbidity in patients living with HIV/AIDS. Indeed, nearly all HIV-infected individuals will develop either oral pharyngeal (OPC) or esophageal candidiasis (EC) at some point in their lives. The ability of C. albicans to cause disease is associated with a morphogenetic transition between round yeast form and filamentous hyphal forms. Consequently, this process has been the subject of intense study and many genes have been identified that affect the yeast-to-filament transition. Less understood is how these genes and regulatory pathways interact to orchestrate this complex biological process. This is due, in part, to the fact that strategies for large-scale genetic interaction screening had not been developed in C. albicans prior to the initiation of our work. We have developed an approach to large scale genetic interaction screening in C. albicans based on the concept of complex haploinsufficiency and applied it to the study of the RAM network (Regulation of Ace2p and Morphogenesis) in morphogenesis. In this way, we found that the RAM pathway interacts with the cAMP/protein kinase A (PKA) pathway to regulate the transcription of a common set of genes during morphogenesis. In this application, we propose to determine the mechanism through which the activities of the RAM and PKA pathways are coordinated (Aim 1). We will also use our collection of mutants to investigate how the RAM pathway functions independently of its well characterized transcription factor Ace2 during serum- induced morphogenesis (Aim 2). Finally, we will develop a library of bar-coded transcription factor deletion cassettes and apply them to a directed complex haploinsufficiency screening approach to more completely define the genetic interaction networks for the RAM and PKA pathways (Aim 3). These screens will be carried out in vitro and using in vivo mouse models of candidiasis and, as such, will represent the first in vivo genetic interaction screen. PUBLIC HEALTH RELEVANCE: Nearly every person living with HIV/AIDS will develop oropharyngeal or esophageal candidiasis during their lifetime. The ability of Candida albicans, the most common human fungal pathogen, to cause mucosal disease is associated with its ability to undergo a morphological change from round yeast form to filamentous hyphal forms. The project proposed in this application is based on the use of a novel genetic approach to develop a deeper understanding the cellular regulatory networks that control this process. Understanding the mechanisms of this process and its relation to pathogenesis could lead to novel approaches to treating or preventing these infections.